During the present reporting period, significant progress was made on this research project. The existence of cannabinoid CB2 receptors in the brain has been heretofore controversial. Most evidence has heretofore suggested that only CB1 cannabinoid receptors are found in brain and central nervous system, while cannabinoid CB2 receptors are restricted to the body's periphery - primarily in the immune system. However, this view has been challenged by recent claims that CB2 receptors are present in the central nervous system and by recent claims that CB2 receptors modulate synaptic activity. Therefore, we used highly selective CB2 agonists and antagonists, combined with the use of CB1 and CB2 receptor gene-deleted mice, and molecular neurobiology techniques combined with electrophysiology, to study the existence and function of CB2 receptors in the brain. Firstly, we studied the expression of functional cannabinoid CB2 receptors on dopamine neurons within the ventral tegmental area (VTA) in rats. The rationale for this work was that we had previously reported the expression of functional cannabinoid CB2 receptors in midbrain dopamine neurons in mice. However, little was known as to whether CB2 receptors are similarly expressed in rat brain. We used in situ hybridization and immunohistochemical assays, and detected CB2 gene and receptors in dopamine neurons of the VTA. The CB2 receptors on VTA dopamine neurons were up-regulated by cocaine self-administration. Electrophysiological experiments showed that activation of CB2 receptors by the CB2-selective receptor agonist JWH133 inhibited VTA dopamine neuronal firing in single dissociated neurons. Local administration of JWH133 by micro-injection into the nucleus accumbens inhibited cocaine-enhanced extracellular dopamine and intravenous cocaine self-administration. This effect was blocked by AM630, a selective CB2 receptor antagonist. These data suggest that CB2 receptors are expressed on VTA dopamine neurons and functionally modulate dopaminergic neuronal activity and cocaine self-administration behavior in rats. We then studied mechanisms underlying cannabis-induced aversion. Cannabis can be rewarding or aversive. Cannabis reward is believed to be mediated by activation of cannabinoid CB1 receptors (CB1Rs) on GABAergic neurons that disinhibit dopaminergic neurons in the ventral tegmental area (VTA). However, little is known about the mechanisms underlying cannabis-induced aversion. We found CB1Rs not only on VTA GABAergic neurons, but also on VTA glutamatergic neurons that express vesicular glutamate transporter 2 (VgluT2). We then used Cre-Loxp transgenic technology to selectively delete CB1Rs in VgluT2-expressing glutamatergic neurons (VgluT2-CB1-/-) and Cre-dependent viral vector to express light-sensitive channelrhodopsin-2 into VTA glutamatergic neurons. We found that photoactivation of VTA glutamatergic neurons produced robust intracranial self-stimulation (ICSS) behavior, which was dose-dependently blocked by DA receptor antagonists, but enhanced by cocaine. In contrast, 9-tetrahydrocannabinol (9-THC), the major psychoactive component of cannabis, produced dose-dependent conditioned place aversion and a reduction in the above optical ICSS in VgluT2-cre control mice, but not in VgluT2-CB1-/- mice. These findings suggest that activation of CB1Rs in VgluT2-expressing glutamate neurons produces aversive effects that might explain why cannabinoids are generally not rewarding in rodents and might also account for individual differences in the hedonic effects of cannabis in humans. We then studied CB2 receptors on dopamine neurons. We generated Cnr2-floxed mice that were crossed with DAT-Cre mice, in which Cre-recombinase expression is under dopamine transporter gene (DAT) promoter control to ablate the Cnr2 gene in midbrain DA neurons of DAT-Cnr2 conditional knockout (cKO) mice. Using novel sensitive RNAscope in situ hybridization, we detected CB2R mRNA expression in VTA DA neurons in wildtype and DAT-Cnr2 cKO heterozygous but not in the homozygous DAT-Cnr2 cKO mice. We found that the deletion of CB2Rs in dopamine neurons enhances motor activities, modulates anxiety and depressive-like behaviors and reduces the rewarding properties of alcohol. Our data reveal that CB2Rs are involved in the tetrad assay induced by cannabinoids which had previously been attributed solely to CB1R agonism. GWAS studies indicate that the CNR2 gene is associated with Parkinson's disease and substance use disorders. Our results suggest that CB2Rs in dopaminergic neurons may play important roles in the modulation of psychomotor behaviors, anxiety, depression, and pain sensation and in the rewarding effects of alcohol and cocaine. We then studied glutamate-cannabinoid interactions in the nucleus accumbens. Metabotropic glutamate receptor 5 (mGluR5) antagonism inhibits cocaine self-administration and reinstatement of drug-seeking behavior. However, the cellular and molecular mechanisms underlying this action are poorly understood. We found that a presynaptic glutamate/cannabinoid mechanism may underlie this action. Systemic or intra-nucleus accumbens (NAc) administration of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) dose-dependently reduced cocaine (and sucrose) self-administration and cocaine-induced reinstatement of drug-seeking behavior. The reduction in cocaine-taking and cocaine-seeking was associated with a reduction in cocaine-enhanced extracellular glutamate, but not cocaine-enhanced extracellular dopamine (DA) in the NAc. MPEP alone, when administered systemically or locally into the NAc, elevated extracellular glutamate, but not DA. Similarly, the cannabinoid CB1 receptor antagonist, rimonabant, elevated NAc glutamate, not DA. mGluR5s were found mainly in striatal medium-spiny neurons, not in astrocytes, and MPEP-enhanced extracellular glutamate was blocked by a NAc CB1 receptor antagonist or N-type Ca++ channel blocker, suggesting that a retrograde endocannabinoid-signaling mechanism underlies MPEP-induced glutamate release. This interpretation was further supported by our findings that genetic deletion of CB1 receptors in CB1-knockout mice blocked both MPEP-enhanced extracellular glutamate and MPEP-induced reductions in cocaine self-administration. Together, these results indicate that the therapeutic anti-cocaine effects of mGluR5 antagonists are mediated by elevation of extracellular glutamate in the NAc via an endocannabinoid-CB1 receptor disinhibition mechanism. We then studied CB1R neutral antagonism. Recent preclinical studies suggest that the neutral CB1R antagonist AM4113 may retain the therapeutic anti-addictive effects of SR141716A in nicotine self-administration models with fewer unwanted side effects. However, little is known about whether AM4113 is also effective for other drugs of abuse, such as opioids and psychostimulants, and whether it produces depressive side effects similar to SR141716A in experimental animals. We showed that systemic administration of AM4113 (3 and 10mg/kg) dose-dependently inhibited self-administration of intravenous heroin but not cocaine or methamphetamine, whereas SR141716A (3 and 10mg/kg) dose-dependently inhibited self-administration of heroin and methamphetamine but not cocaine. In the electrical brain-stimulation reward (BSR) paradigm, SR141716A dose-dependently decreased brain-stimulation reward, but AM4113 had no such effect. We suggest that CB1 neutral antagonists may constitute a new and useful groiup of anti-addiction medications.